Annexin V-FITC / PI Apoptosis Reagents

Apoptosis, or programmed cell death, is a natural process of cellular self-destruction. Apoptosis is necessary for routine cell turnover and tissue homeostasis. It is also important in embryogenesis, maintenance of immune tolerance, and development of the nervous system. Problems with the regulation of apoptosis are thought to be linked to many cancers, degenerative diseases, and autoimmune diseases, making apoptosis a key target in many fields of clinical research.

Figure 1. Scatter plots following Cellometer Vision CBA Analysis show an increase in the apoptotic cell population from 4% to 22% following incubation with 10µm α-Tos, an apoptosis-inducing drug.

Principle of the Annexin V-FITC / PI Apoptosis Assay

Annexin V is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS). PS is normally only found on the intracellular leaflet of the plasma membrane in healthy cells, but during apoptosis, PS translocates to the external leaflet. Fluorochrome-labeled Annexin V can then be used to specifically target and bind the PS on the surface of apoptotic cells. Cells undergoing necrosis following tissue damage or disease have compromised membranes that will also allow binding of PS. An additional dye is utilized to help differentiate necrotic cells within the population.

Propidium Iodide (PI) is a membrane-exclusion dye that permeates cells with compromised cell membranes and binds to DNA. Early apoptotic and healthy cells with intact membranes will exclude PI, while very late stage apoptotic and necrotic cells with compromised membranes are stained. Use of both Annexin V-FITC and PI allows researchers to characterize a cell population based on % normal, % apoptotic, and % necrotic /very late-stage apoptotic cells.

ViaStain™ Live Caspase 3/7 Detection for 2D/3D Culture with and without Hoechst

ViaStain™ Live Caspase 3/7 Detection for 2D/3D Culture kit is designed to detect caspase 3/7 activity in live cells. The ability to perform kinetic apoptosis assays allows researchers to continuously measure the caspase 3/7 activity within the cell population. This no wash assay has been shown to effectively work in both 2D and 3D cultures.

The reagent (NucViewTM) consists of a nucleic acid-binding dye with a fluorescent probe that is attached to a four-amino acid peptide sequence DEVD (Asp-Glu-Val-Asp) forming a cell membrane-permeable DEVD-DNA complex. While the nucleic-acid dye is linked to the DEVD peptide sequence, the dye is unable to bind to DNA and remains non-fluorescent. During apoptosis, caspase 3/7 proteins cleave the DEVD-DNA dye complex and thereby release the high-affinity DNA dye, which translocates to the nucleus and binds to the DNA, producing a bright green fluorescent signal.

To enumerate the total number of nucleated cells and obtain percent of apoptotic cells, the cells can be counterstained with Hoechst 33342.

Caspase-3 Apoptosis Kit

Caspase-3 belongs to a family of evolutionally conserved cysteine proteases that play a key role in regulating programmed cell death, or apoptosis. Caspase-3 activation triggers a series of irreversible events that lead to the death of the cell, including activation of the CAD endonuclease that degrades DNA within the nucleus and initiates chromatin condensation. Increasing evidence suggests involvement of caspase-3 in Alzheimer’s Disease (AD) via cleavage of amyloid prescursor protein (APP) and tau and the formation of beta-amyloid (Aβ) and neurofibrillary tangles (NFTs). Other studies have demonstrated a role for caspase-3 in growth stimulation via PGE2. High amounts of caspase-3 have been linked to an increased rate of cancer recurrence and death in patients undergoing radiation treatment through the stimulation of growth of surviving cancer cells, making caspase-3 a key prognostic marker and therapeutic target in cancer therapy.

CaspGLOW™ Fluorescein in Active Caspase-3 Staining Kit

Caspase-8 Apoptosis Kit

The Cellometer Caspase-8 Apoptosis Kit is designed for fast, simple determination of Caspase-8 activity for a cell population using image cytometry. Each kit contains ready-to-use reagents sufficient for 100 tests along with a detailed instruction manual outlining cell staining, imaging, and cell cycle data analysis using the Cellometer Vision CBA Analysis System.

Principle of the Cellometer Caspase-8 Apoptosis Assay

The CaspGLOW™Fluorescein Active Caspase-8 Staining Kit offers a simple, sensitive method for detection of active Caspase-8 in living cells. When detecting caspase-8, we utilize a specific FITC-conjugated caspase-8 inhibitor, IETD-FMK. IETD-FMK is cell-permeable and binds irreversibly to active caspase-8 within the cell. The fluorescent marker (FITC) on the IETD-FMK allows for the detection of cells undergoing apoptosis via Caspase-8 activation using the Vision CBA Analysis System. Results are presented as a Caspase positive population percentage and cell concentration.